Metastatic breast cancer is incurable with present therapy. Immunotherapy using autologous dendritic cells to stimulate tumor-specific CD8+ and CD4+ T cells offers a new avenue in the treatment of individuals with metastatic disease. In the past year, we have initiated a phase I/II trial designed to test the effectiveness of administering dendritic cells (DCs) pulsed with an altered peptide ligand from the HER-2/neu protein to women with metastatic breast cancer. Although only two patients have received at least three vaccinations, we have been encouraged that one of these patients has had a significant partial response to this treatment. In this project, we have brought together experts in the areas of structural immunology, dendritic and T cell immunology, and viral vaccines, to design a more comprehensive HER-2/neu-based vaccine. We will investigate in vitro and in vivo using animal models whether altering multiple epitopes from the HER-2/neu protein can induce a more significant T cell and anti-tumor effect compared to the single altered epitope in our current clinical trial. Additionally, we will perform preclinical work in vitro using human DCs and in vivo using transgenic mice, to test the effectiveness of a novel viral system, Venezuelan equine encephalitis replicon particles (VRP), with a natural tropism for DCs to target the entire HER-2/neu gene and cytokines to DCs. We will evaluate in transgenic mice whether the anti-tumor and T cell activity of DCs infected with VRP is greater than that found using peptide-pulsed DCs. In year four of this proposal, we will use the data generated from these studies to design a second-generation vaccine with broader anti-tumor and T cell activity to be used in the next clinical trial at the Lineberger Cancer Center.